The Genome-wide Resequencing Analysis Of A Yellow-leaf Lotus Japonicus Mutant Induced By Intermediate-energy Carbon Ion Beam Irradiation

Carbon ion beam is an excellent physical mutagen,which has been widely used in mutation breeding and functional genomics research in recent years.After carbon ion beam irradiation on Lotus japonicus and mutation screening,a yellow-leaf mutant?named C16?from M₂ generation was obtained.Through several generations of genetic stability verification,a stable C16 mutant from M₄ generation was finally obtained.?1?In contrast to healthy mature Lab-WT plants,the C16 mutant plants manifested very different phenotypes,including dwarf,yellower,smaller leaves as well as the mature leaves were lighter and yellower.Furthermore,the growth and development of C16 plants were retarded.?2?The photosynthetic physiological parameters of mature leaves of a series of Lab-WT and C16 healthy plants were measured,including chlorophyll content?the total,chlorophyll a and chlorophyll b?and chlorophyll fluorescence parameters Fv/Fm?maximum photochemical efficiency of PSII?.The results of chlorophyll content determination showed that the total chlorophyll content,chlorophyll a content and chlorophyll b content in mature leaves of C16 were significantly lower than those of Lab-WT.The lower chlorophyll content of mature leaves of C16 changed the proportion of pigments and pigment content in leaves.It is speculated that the lower chlorophyll content may be responsible for the yellow-leaf phenotype of the mutant.To discover the differences in photosynthetic capacity of mature leaves of healthy Lab-WT and C16 plants,the chlorophyll fluorescence technique was used to determine the parameters Fv/Fm,which reflects the potential photosynthetic ability.The Fv/Fm of mature leaves of C16 plants was significantly lower than that of Lab-WT,suggesting that the photosynthetic capacity of C16 mature leaves was decreased than that of Lab-WT.Combined with previous results,the photosynthetic capacity of C16 mature leaves was significantly decreased than that of Lab-WT,it is easy to infer that lower chlorophyll content of C16 mature leaves is the main cause.?3?To explore the molecular mechanisms of C16 mutant,the whole genome re-sequencing techniques were used to re-sequence whole genomes of both healthy Lab-WT and C16 mature leaves.After resequencing analysis,95 SBSs and 17 small INDELs were found in the C16 mutant genome.Through SnpEff annotation,it was found that the distributions of the 112 mutation loci in the different structures of the genes were located unevenly,with the most mutation sites in the upstream and downstream regions.In all the chromosomes?Chr?,most of mutation sites were located in Chr1,including38 SBSs and 6 small INDELs;the least proportion of mutation sites was located in Chr5,including 5 SBSs and 1 small INDELs.Through annotation of 112 mutation loci,78mutated genes was annotated with SnpEff,among which there was one Lj5g3v1669360.1 gene with the SBSs mutation site C5-TG,belonging to the AGPs coding family gene.AGPs encoding gene family is related to chlorophyll synthesis and leaf color.Therefore,gene Lj5g3v1669360.1 was speculated as the candidate gene resulting in the mutation phenotypes in C16 mutant.?4?Using qRT-PCR technique,the differential expression level of gene Lj5g3v1669360.1 in mature leaves of Lab-WT and C16 was analyzed.The results showed that the expression level of Lj5g3v1669360.1 in mature leaves of C16 was significantly lower than that of Lab-WT.Therefore,the result might reveal the reason why the phenotypes of lower chlorophyll content,weaker photosynthetic ability,yellower leaves and slower growth were observed in C16 mutant.